Rapid methods for identifying modifiers of cellular apoptosis activity

ABSTRACT

The invention provides a single-well, microscale method of determining the specific apoptotic activity of a cell. The method consists of contacting a cell population of about 1×10 5  cells for a time period of between about 30 minutes and 4 hours with a sufficient volume of medium containing an apoptotic specific diagnostic reagent and a diagnostic accessory reagent so as to cover the cell population, and determining the activity of the apoptotic specific diagnostic reagent. The invention also provides a method of identifying a compound which induces apoptosis. The invention further provides a rapid method of identifying a compound which inhibits apoptosis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of pending U.S. patent application Ser.No. 08/869,553, filed Jun. 5, 1997.

BACKGROUND OF THE INVENTION

This invention relates to the biological process of programmed celldeath and, more specifically to rapid methods of measuring apoptoticactivity and of screening for compounds which modulate apoptosis.

Cells can die by at least two fundamentally different biologicalprocesses. One process, termed necrosis, refers to cell or tissue deathwhich usually occurs as a result of massive physical or chemical insult.Necrosis is characterized, in part, by cell swelling organelledisintegration and the leakage of cell cytoplasm into the extracellularspace and is generally considered to be a passive process.

The alternative process of cell death is known as apoptosis orprogrammed cell death. This mechanism of cell death in mammalian cellsis characterized by a set of morphological and biochemical changes thatreflect an active cell suicide. Apoptotic changes include cellshrinkage, nuclear chromatin condensation and margination, and DNAfragmentation. Biochemical events include the externalization ofphosphatidyl serine and the activation of aspartate-specific cysteineproteases.

In regard to the latter biochemical event, proteases within the cysteineaspartic acid protease family, also known as the ICE/CED-3 family, arecritical for effecting the process of apoptosis. These enzymes arecysteine proteases and exhibit substrate specificity for cleavage afteran aspartic acid residue. Due to these characteristics, these enzymesare now referred to by the above term “cysteine aspartic acid proteases”or “caspases”. During the apoptotic process, caspase activity isgenerated in cells and known inhibitors of the caspase family ofproteases inhibit apoptosis.

Apoptosis is important clinically for several reasons. In the field ofoncology, many of the clinically useful drugs kill tumor cells byinducing apoptosis. For example, cancer chemotherapeutic agents such ascisplatin, etoposide and taxol all induce apoptosis in target cells. Inaddition, a variety of pathological disease states can result from thefailure of cells to undergo proper regulated apoptosis. For example, thefailure to undergo apoptosis can lead to the pathological accumulationof self-reactive lymphocytes such as that occurring in many autoimmunediseases, and can also lead to the accumulation of virally infectedcells and to the accumulation of hyperproliferative cells such asneoplastic or tumor cells. The development of efficacious compoundswhich are capable of specifically inducing apoptosis would therefore beof therapeutic value in the treatment of these pathological diseasesstates.

In contrast, the inhibition of apoptosis is also of clinical importance.For example, cells are thought to die by apoptosis in the brain andheart following stroke and myocardial infarction, respectively.Moreover, the inappropriate activation of apoptosis can also contributeto a variety of other pathological disease-states including, forexample, acquired immunodeficiency syndrome (AIDS), neurodegenerativediseases and ischemic injuries other than those listed above. Asapoptotic inducers are of benefit in the previously mentioned diseasestates, specific inhibitors of apoptosis would similarly be oftherapeutic value in the treatment of these latter pathological diseasestates.

Drug discovery benefits from the use of efficient high throughputmethods which can rapidly identify specific molecules that interact withthe target of interest. The identification of compounds whichspecifically modulate the apoptotic pathway so far has been hindered bythe lack of such methods. Available methods are either limited by thelack of specificity and/or efficiency. For example, most anti-cancerdrugs are screened for their ability to kill cells and therefore willidentify compounds that induce both necrosis or apoptosis. Moreover,many of these methods are often cumbersome in that they requireassessment of cell viability and take days to perform.

Attempts have been made to create methods that are specific forapoptosis. For example, the amount of DNA degradation induced in a cellpopulation has been used for a measurement of apoptosis. However, DNAdegradation is a relatively late step in the apoptotic process.Moreover, DNA degradation is not truly specific for apoptosis since DNAeventually becomes degraded in necrotic cells too.

Other methods currently employed use metabolic determinations as anattempt to measure apoptosis in a relatively shorter time frame. Forexample, cells undergoing apoptosis show impaired mitochondrial functionwhich can be measured using dyes such as alamar blue or by colourimetricassays such as reduction of MTT (3-(4.5-dimethyl)thiazol-2-yl-2,5-diphenyl tetrazolium bromide) to formazan. However,impaired mitochondrial function is not specific for apoptosis as it isalso a characteristic exhibited by necrotic cells.

There has been one method described where apoptotic measurements appearspecific and are conducted in relatively short time periods. Forexample, the successful measurement of caspase activity by measuring thefluorescent cleavage product of the CPP32 substrate analog DEVD-AMC (SEQID NO:1) has been reported (Armstrong et al. J. Biol. Chem.271:16850-16855 (1996)). However, this method required separatepreparations of the cell lysate and reaction mixture as well asadditional manipulations, including sample washing in the assayprocedure. In addition to the extra time required to perform theseadditional manipulations, this method could not be performed in a singlestep due to the requirement for separate preparation of lysate andreaction mixture. Therefore, regarding high throughput assays forapoptosis, what benefit might have been gained in specificity was lostdue the inefficiencies incurred in order to measure the caspaseactivity.

Another method specific for apoptosis has been reported where separatepreparations of cell lysate and reaction mixture has not been required(Los et al. Nature 375:81-83 (1995)). This method similarly determinedthe caspase activity following induction of apoptosis by measuring thecleavage product of an ICE substrate analogue. Separate preparation ofsamples and reaction was avoided due to the use of a detergent whichdoes not completely lyse the cells. However, the incompletesolubilization of cellular components can result in decreasedsensitivity of the method. Moreover, the detection of substrate cleavagewas performed by a separate procedure and, as with the method ofArmstrong et al. above, similarly required additional manipulations andsteps which lengthened the time period for the procedure.

Thus, there exists a need for rapid and efficient methods to identifycompounds which can specifically modulate the apoptotic pathway for thetherapeutic treatment of human diseases. The present invention satisfiesthis need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a single-well, microscale method of determiningthe specific apoptotic activity of a cell. The method consists ofcontacting a cell population of about 1×10⁵ cells for a time period ofbetween about 30 minutes and 4 hours with a sufficient volume of mediumcontaining an apoptotis-specific diagnostic reagent and a diagnosticaccessory reagent so as to cover the cell population, and determiningthe activity of the apoptotis-specific diagnostic reagent. The inventionalso provides a method of identifying a compound which inducesapoptosis. The method consists of (a) providing a cell over-expressing acell survival polypeptide at a level which is sufficient to prevent theinduction of apoptosis; (b) treating the cell over-expressing the cellsurvival polypeptide with a direct stimulus of the cell death pathway;(c) adding a compound to be tested for apoptotic inducing activity, and(d) determining cellular apoptotic activity, the presence of which isindicative of the compound being an apoptotic inducer. The inventionfurther provides a rapid method of identifying a compound which inhibitsapoptosis. The method consists of (a) separately contacting a pluralityof cell populations with a different compound to be tested for apoptoticinhibiting activity; (b) incubating the cells with a direct stimulus ofthe cell death pathway for a period of between about 2 minutes to 3hours, and (c) measuring the specific apoptotic activity of the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the single-well method of measuringspecific apoptotic activity with a method requiring additional washingprocedures.

FIG. 1A shows the caspase activity induced in neo-transfected Jurkatcells in the presence (closed symbols) or absence (open symbols) ofanti-Fas antibody.

FIG. 1B shows the inhibition of induced caspase activity in Bcl-2transfected cells in the presence or absence of anti-Fas anti-body. (:wash+anti-Fas; ∘:wash-anti-Fas; ▪:single-well+anti-Fas;□:single-well-anti-Fas).

FIG. 2 shows the induced caspase activity measured at different timepoints following treatment with a range of anti-Fas antibodyconcentrations.

FIG. 2A shows the anti-Fas antibody titration and caspase time course inneo-transfected Jurkat cells.

FIG. 2B shows a parallel titration and time course in Bcl-2 transfectedJurkat cells.

FIG. 3 shows the screening of a chemical compound library using Bcl-2transfected cells primed with anti-Fas antibody and the identificationof positive apoptotic inducers.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a novel method for the rapid and efficientmeasurement of apoptosis. The method is advantageous in that itdistinguishes apoptosis from other forms of cell death and can beperformed in a single step and on a small scale analysis. Moreover, themethod is rapid in that it can be performed in a time period of betweenabout 30 minutes and 4 hours. These advantages allow for the highthroughput screening of a large number of samples with a specificity andtime period not achieved before with previous methods known in the art.The method distinguishes apoptosis from other forms of cell death inthat it measures the activity of cysteine aspartic acid proteases orother specific indicators of apoptosis. Therefore, the method can beadvantageously used for the specific identification of inducers ofapoptosis such as compounds that inhibit the function of Bcl-2.

In one embodiment, the invention is directed to a method of measuringapoptosls where a single buffer is added which contains a lysis agentand a detectable substrate for one or more aspartate specific cysteineproteases. A specific example of a detectable substrate is the peptideanalogue DEVD-AMC which fluoresces after protease cleavage. Simultaneouslysis with substrate allows for the rapid measurement of aspartatespecific cysteine protease activity without the necessity for washes orother transfer of reagents.

In another embodiment, the invention is directed to a method foridentifying compounds which induce apoptosis. The method employs themeasurement of induced cysteine aspartic acid protease activity in acell line that expresses the Fas antigen and has been engineered tooverexpress the cell survival polypeptide Bcl-2 and which has beentreated with anti-Fas antibody. In a normal unengineered cell line,treatment with the anti-Fas antibody causes rapid apoptosis. However, inthe engineered cell line apoptotic progression is blocked due to thecontinued presence of Bcl-2. Compounds that induce apoptosis areidentified by incubating them with the engineered cell line that hasalso been incubated with anti-Fas antibody and then measuring apoptosisusing the method described above. Due to the overexpression of Bcl-2,this method for identifying inducers of apoptosis will identifycompounds that either inhibit the function of Bcl-2, or that stimulatethe cell death pathway downstream of the Bcl-2 blockade. Moreover, sincethe engineered cells are treated with the pro-apoptotic anti-Fasantibody, these cells are primed for programmed cell death so that thereis little or no lag time once the cells are treated with a positiveapoptotic inducer. This priming of the cells provides additionalsensitivity and speed for identifying apoptotic inducers. The primingfor cell death can be provided by other pro-apoptotic stimuli includingbut not limited to, staurosporine, TNF and TNF plus cycloheximide.

In another embodiment, the invention is directed to a method ofidentifying inhibitors of apoptosis. This method also utilizes thesingle-step method described above, however, the indicator cell linedoes not require the overexpression of a cell survival polypeptide.Instead, the cells are first incubated with a compound to be tested forinhibitory activity and then treated with an direct stimulus ofapoptosis such as anti-Fas antibody. In the absence inhibitorycompounds, cells subsequently treated with anti-Fas antibody undergoapoptosis and exhibit a rapid induction of aspartate specific cysteineprotease activity. In contrast, positive compounds inhibit the emergenceof protease activity compared to control samples.

As used herein, the term “apoptosis” is intended to mean thephysiological process known as programmed cell death. This process is amorphologically and biochemically distinct form of cell death thatregulates cell turnover under normal physiological conditions. Themorphological features include an orchestrated sequence of changes whichinclude cell shrinkage, chromatin condensation, nuclear segmentation andeventual cellular disintegration into discrete membrane-bound apoptoticbodies. The biochemical features include, for example, internucleosomalcleavage of cellular DNA and the activation of ICE/Ced-3 family ofproteases. The term “apoptosis” is used here synonymously with thephrase “programmed cell death.” These terms are intended to beconsistent with their use as they are known and used by those skilled inthe art.

As used herein, the term “single-well” when used in reference to anapoptotic assay method is intended to mean that all actions, processes,or measures taken to achieve the determination of specific apoptoticactivity are performed without the need for intermediary processingsteps that require the cells to be moved from one well or vessel toanother. Such intermediary processing steps can include, for example,cell harvesting; washing steps; purification steps; separation steps;exchange of additional buffers or reagents; media, reagent or lysatetransfer; or, sample transfer or processing for analysis. The termincludes, however, the addition or mixing of other buffers or reagents.Thus, the term “single-well” is intended to mean that the method can beperformed by adding to a cell sample all reagents necessary to determinethe specific apoptotic activity following an appropriate time ofincubation. The addition of such reagents can be performed in a singlestep or they can be added sequentially. Measurement of specificapoptotic activity therefore occurs without further processing ortransfer of the sample. The term single-well is also intended to includescalable formats and automated procedures.

As used herein, the term “microscale” is intended to mean that themethod can be performed on a scale measured in microliter (μl) orsub-microliter volumes and whereby many sample measurements can be madein parallel in, for example, multi-well plates. Such a scale is incontrast to milliters (ml) and to procedures not amenable to amulti-well format. A specific example of a multi-well format is a96-well ELISA plate. The microscale measurable volumes are between about1 and 200 μl, more preferably between about 30 and 125 μl, andpreferably about 100 μl. Thus, a microscale method can be entirelyperformed, for example, in a microwell plate format such as a 96 well orother multiwell sample format.

As used herein, the term “specific apoptotic activity” is intended tomean cellular activity specifically due to the activation of theprogrammed cell death pathway. Programmed cell death is effected andregulated by a variety of molecules including the Bcl-2 and Baxpolypeptides as well as the activation of members of the ICE/Ced3-familyof cysteine aspartic acid proteases (caspases). Thus, cellular activityspecifically due to the activation of the programmed cell death pathwayis intended to mean the activity of molecules, including the abovefamilies of molecules, which regulate or participate in the apoptosispathway and whose activity correlates with the appearance of apoptosisin these cells.

As used herein, the term “direct stimulus” when used in reference to thecell death pathway is intended to mean an agent that increases thespecific apoptotic activity of a cell. Specific examples of directstimuli include, for example, Fas ligand, anti-fas antibody,staurosporine, ultraviolet (UV) and gamma irradiation. Other directstimuli exist and are known by those skilled in the art. Thus, a directstimulus of apoptosis is an agent which increases the molecular activityof the above-described families of molecules which enhance orparticipate in apoptosis.

As used herein, the term “apoptotic specific diagnostic reagent” or“diagnostic reagent” is intended to mean a reagent which specificallymeasures or can be made to specifically measure the specific apoptoticactivity of a cell. Such reagents include, for example, the measurementof caspase activity. Moreover, such measurements can be either direct orindirect. Apoptotic specific diagnostic reagents can include substrateanalogues for caspase family members which fluoresce upon cleavage.Specific examples of such substrate analogues include, for example,ZEVD-AMC, YVAD-AMC and DEVD-AMC(carbobenzoxy-Glu-Val-Asp-aminomethylcoumarin, Tyr-Val-Ala-Asp-AMC (SEQID NO:2) and Asp-Glu-Val-Asp-AMC (SEQ ID NO:1), respectively).

Reagents other than those which measure the binding or activity of cellsurvival or cell death polypeptides are similarly included within thedefinition of the term so long as such reagents can specificallymeasure, or be made to specifically measure apoptotic events. An exampleof such an apoptotic specific diagnostic reagent is the phospholipidbinding polypeptide Annexin V. This reagent binds phosphatidylserine ina calcium dependent manner. Specificity of phosphatidylserine as ameasure for apoptotic cells is due to the fact that this lipid isprimarily found on the inner surface of a cell membrane but istranslocated to the outer surface upon cell death. Its use inconjunction with a viability stain can provide specificity for apoptoticmediated cell death. Thus, the measurement of phosphatidylserine on theouter membrane surface can be used as an apoptotic specific diagnosticreagent. Other specific indicators of apoptosis exist and are known tothose skilled in the art. Such other specific indicators are intended tobe include within the definition of the term as defined and used herein.

As used herein, the term “diagnostic accessory reagent” or “accessoryreagent” is intended to mean an agent that is required for the apoptoticspecific diagnostic reagent to function. The agent can be a single ion,an organic or inorganic molecule, a macromolecule or any combinationthereof. The requirement for function of the diagnostic reagent can bebiochemical or physical. A specific example of a biochemical requirementis found with the case of Annexin V where calcium is necessary forphospholipid binding. A physical requirement for an accessory reagent isin the case where caspase activity is measured. In order to measurecaspase activity, either the cell has to be lysed or the substrateanalogue has to permeate the membrane. The accessory reagent wouldfacilitate such a lysis or crossing of the membrane. Specific examplesof such accessory reagents would be an agent which is capable of lysingthe cell or disintegrating the cell membrane, or alternatively, an agentwhich punctures holes in the membrane to allow for molecules andmacromolecules to cross the membrane. Such agents are known to thoseskilled in the art and are intended to be within the scope of thedefinition as it is used herein.

The term “lysis reagent” as it is used herein is therefore intended tomean a reagent which is capable of causing the dissolution ordestruction of cellular membrane integrity. Such reagents generallyinclude, for example, detergents such as SDS, NP-40 and triton, however,lysis reagents other than detergents are also included within thedefinition so long as such reagents do not interfere with the cellularapoptotic activity and detection using an apoptotic specific diagnosticreagent. Additionally, the meaning of the term is also intended toinclude detergents or other reagents which do not result in cellulardestruction, but instead, are capable of permeabilizing the membrane andallowing entry of the diagnostic reagent. A specific example, of such alysis reagent includes digitonin.

As used herein, the term “compound” when used in reference to an agentcapable of inducing or inhibiting apoptosis is intended to mean amolecule able to modulate the specific apoptotic activity of a cell.Such a molecule can include small organic or inorganic molecules as wellas large macromolecules. Specific examples of small molecules includeetoposide and carbobenzoxy-Val-Ala-Asp-fluoromethylketone. Examples ofmacromolecules which are able to modulate the specific apoptoticactivity of a cell include peptides, polypeptides, proteins, nucleicacid, carbohydrate and lipid. Functional or structural analogues ormimics of such compounds which exhibit substantially the same activationor inhibition activity are also included within the meaning of the termas used herein. The type, size or shape of the molecule is not importantso long as the molecules can either induce or inhibit the specificapoptotic activity of a cell.

As used herein, the term “cell survival polypeptide” is intended to meana peptide, polypeptide or protein that is capable of inhibiting thespecific apoptotic activity of a cell. Cell survival polypeptidesincludes those polypeptides which directly regulate the programmed celldeath pathway such as Bcl-2, Bcl-xL, Mcl-1 and the ElB-19K protein ofadenovirus as well as those which indirectly regulate the programmedcell death pathway.

The meaning of the term “cell survival polypeptide” also includesfunctional fragments so long as they have the ability to inhibit thespecific apoptotic activity of a cell. The term is also intended toinclude polypeptides that include, for example, modified forms ofnaturally occurring amino acids such as D-steroisomers, non-naturallyoccurring amino acids, amino acid analogues and structural mimics solong as such polypeptides retain functional activity as defined above.

As used herein, the term “overexpressing” when used in reference to thelevel of a cell survival polypeptide is intended to mean an increasedaccumulation of the cell survival polypeptide in the overexpressingcells compared to their levels in counterpart normal cells.Overexpression can be achieved by natural biological phenomenon as wellas by specific modifications as is the case with genetically engineeredcells. Overexpression also includes the achievement of an increase incell survival polypeptide by both endogenous or exogenous mechanisms.Overexpression by natural phenomenon can result by, for example, amutation which increases expression, processing, transport, translationor stability of the RNA as well as mutations which result in increasedstability or decreased degradation of the polypeptide. Such examples ofincreased expression levels are also examples of endogerious mechanismsof overexpression. A specific example of a natural biologic phenomenonwhich results in overexpression by exogenous mechanisms is the adjacentintegration of a retrovirus. Overexpression by specific modification canbe achieved by, for example, the use of recombinant methods known in theart to construct and overexpress stably or transiently a cell survivalpolypeptide in a compatible vector-host system.

As used herein, the term “cell death polypeptide” is intended to mean apeptide, polypeptide, or protein that is capable of increasing orinducing the specific apoptotic activity of a cell. Cell deathpolypeptides includes those polypeptides which directly regulate theprogrammed cell death pathway such as Bax, Bad, Bcl-xS, Bak, and Bik aswell as those which indirectly regulate the programmed cell deathpathway. Cell death polypeptides also includes, for example,ICE/Ced3-family of caspases since such caspases can induce programmedcell death.

The meaning of the term “cell death polypeptide” also includesfunctional fragments so long as they have the ability to enhance orinduce the specific apoptotic activity of a cell. As with functionalfragments of the counterpart cell survival polypeptides, the term isalso intended to include polypeptides that include, for example,modified forms of naturally occurring amino acids such asD-steroisomers, non-naturally occurring amino acids, amino acidanalogues and structural mimics so long as such polypeptides retainfunctional activity as defined above.

As used herein, the term “aspartate specific cysteine protease” or“caspase” is intended to mean those family of proteases which arerelated genetically to the C. elegans ced-3 gene product and includes,for example, human ICE (interleukin-1-β converting enzyme), ICH-1_(L),CPP32, Mch2, Mch3, Mch4, Mch5, Mch6, ICH-2 and ICE_(rel) ⁻III. Inaddition to the shared homology to Ced3, the known caspases also sharethe following characteristics: 1) they exhibit cysteine proteaseactivity with specificity for substrate cleavage at Asp-x bonds, 2)contain a conserved pentapeptide sequence (QACRG (SEQ ID NO:3), QACGG(SEQ ID NO:4) or a related sequence) within the active site and 3) aresynthesized as proenzymes that require proteolytic cleavage at specificaspartate residues for activation of protease activity. Although theseproteases are defined herein as cell death polypeptides, severalalternative structural forms of caspases exist, such as ICEδ, ICEε,ICH-1_(s) and Mch2β, which function to inhibit apoptosis. Thesealternative forms essentially act as dominant negative mutations andtherefore are considered to be cell survival polypeptides.

The invention provides a single-well, microscale method of determiningthe specific apoptotic activity of a cell. The method consists ofcontacting a cell population of about 1×10⁵ cells for a time period ofbetween about 30 minutes and 4 hours with a sufficient volume of mediumcontaining an apoptotic specific diagnostic reagent and a diagnosticaccessory reagent so as to cover the cell population, and determiningthe activity of said apoptotic specific diagnostic reagent.

The method of the invention provides significant advantages over methodsknown in the art in that it is rapid, efficient and is specific forapoptosis. The speed and efficiency are attributed at least to therequirement of all steps being carried out without removing cells from asingle microwell for the measurement of apoptotic activity. Specificitycan be attributed at least to the measurement of apoptotis-specificbiochemical events.

In regard to the speed and efficiency, separate procedures are notrequired for cell lysis and assay conditions. Moreover, following theappropriate incubation time for specific apoptotic events to occur, themethod is absent of further washing procedures, transfers to additionalcontainers and/or subsequent manipulations required for independentmeasurement procedures. Thus, the method of the invention essentiallytakes a sample of cells and adds a single solution which contains allreagents necessary for the specific detection of apoptosis, incubatesthe mixture and then measures the apoptotic activity within the mixture.

Apoptosis can be measured in a variety of cell types, sources andformats. Usually the cell sample will be, for example, a eukaryotic cellline or other culturable cell type and is plated into a tissue culturedish or multiwell plate treated or untreated to allow analysis of thecell sample. The cell sample can be, for example, an adherent cell typeor one which grows in suspension. Other types of cell samples such astissues are similarly amenable for use in the method of the invention.Those skilled in the art will know, or can determine, the appropriateconditions which are necessary to enable practicing of the method onvarious types of cell samples.

The cell number utilized in the methods of the invention can be varieddepending on the cell type and which diagnostic reagent will be used todetermine specific apoptotic activity of the cell. For example, theJurkat T-cell line expresses relatively high levels of caspase activityand fewer than 100,000 cells are required to obtain an accuratemeasurement. Cells which express lower levels of caspase activitycompared to the Jurkat line can also be used. However, a larger cellpopulation will necessarily be employed. Those skilled in the art willknow or can determine the relative levels of caspase activity in aparticular cell sample and will therefore know the quantity of cellsrequired for determining specific apoptotic activity.

Therefore, the invention provides the measurement of specific apoptoticactivity in a cell population with as few as 10,000 and as many as 1×10⁶cells. Usually the cell population is greater than about 50,000 to allowfor relatively simple detection and preferably the cell population isabout 100,000. Of course, the cell population can be further decreasedcompared to those sizes given above, and the method can be compensatedfor by allowing a slightly longer incubation time with the apoptoticstimulus, detection reagent and/or both. Thus, there are a variety ofalternatives available to those skilled in the art which can be employedto practice the invention.

Reagents necessary for apoptosis include an apoptotic specificdiagnostic reagent and an accessory reagent. The diagnostic reagent willvary depending on the specific apoptotic event that will be measured.For example, caspase activity can be used as a marker of specificapoptotic activity. Activity can be determined using, for example,caspase substrate analogues which contain a detectable moiety. Althougha variety of different labels can be utilized for the detectable moiety,for use in the single-well method of the invention, the detectablemoiety should have a different property following cleavage of substratewhen compared to the starting substrate analogue. The change inproperties for the label allows for measurement of specific apoptoticactivity without the need for additional steps to be performed in theassay to remove, for example, unreacted or unbound substrates fromproducts. Thus, diagnostic reagents which contain detectable moietiesthat fluoresce or luminesce, for example, upon cleavage are amenable foruse in the single-well method of the invention. Specific examples ofsuch caspase substrate analogues containing detectable moieties thatfluoresce upon cleavage include the peptide analogues ZEVD-AMC, YVAD-AMC(SEQ ID NO:2) and DEVD-AMC (SEQ ID NO:1). Alternatively, detectablemoieties which fluoresce at a different wavelength following cleavage oreven following binding to a caspase are also amenable for use in themethods described herein.

Apoptotic specific diagnostic reagents other than caspase substratesexist as well and can alternatively be used in the methods of theinvention. A specific example of such other diagnostic reagents includesthe phospholipid binding polypeptide Annexin V. As with theabove-described caspase substrate analogues, these other diagnosticreagents can similarly be used in the methods of the invention becausethey measure a specific apoptotic event, or can be made to measure aspecific apoptotic event, and also do not require additional proceduresor manipulations in order to obtain a positive indication of apoptoticactivity.

Annexin V, for example, has a high affinity for phosphatidylserine whichis translocated to the outer membrane during cell death. Binding ofAnnexin V is therefore a measure of apoptotic activity. Phospholipidbinding can be determined by, for example, FITC labeled Annexin Vfollowed by removal of unbound label by filtration. The use of samplewells having bottoms comprising membrane filters can be used inconjunction with Annexin V as a diagnostic agent since filtration andsubsequent analysis can occur without the need for sample transfer oradditional processing steps. Diagnostic reagents other than Annexin Vexist and are known to those skilled in the art. Such other reagents cansimilarly be used in the methods of the invention given the teachingsprovided herein.

An accessory reagent is also required for use in the claimed methods ofthe invention. The accessory reagent will vary depending on the type ofdiagnostic reagent used. For example, when using a caspase substrateanalogue as the diagnostic reagent, the accessory reagent will be anagent that allows for the lysis, solubilization or permeabilization ofthe cell membrane. Such procedures will either liberate the cytoplasmiccontents of a cell to allow for measurement of caspase activity or allowtransport of the substrate analogue across the lipid bilayer. Reagentsother than those that destroy or permeabilize the membrane can beemployed as well so long as they allow the colocalization of thediagnostic reagent with the particular molecule or activity to bemeasured. For example, lipid vesicles or other delivery particles aswell as receptor mediated events can similarly be used as the accessoryreagent for colocalizing the diagnostic reagent with the cytoplasmiccontents of an apoptotic cell.

Selection of an accessory reagents will vary depending on the particularapplication and/or apoptotic event that will be measured. Accessoryreagents have been described above in reference to caspase activity forthe measurement of cytoplasmic apoptotic events. However, alternativeaccessory reagents can be employed for the measurement of apoptoticevents on the cell surface or cell exterior. In this regard, it is notnecessary to bring together the cytoplasmic contents and the diagnosticreagent if the apoptotic event is localized on the cell surface orexterior. In this particular case, the accessory reagent can be, forexample, an agent which allows for better access of the diagnosticreagent to the cell surface or exterior.

In addition, accessory reagents can also be, for example, cofactors orother compounds which enable functioning of the diagnostic reagent. Forexample, the diagnostic reagent Annexin V is a calcium dependentphospholipid binding polypeptide. An accessory reagent for Annexin V cantherefore be the presence of calcium in the medium to facilitate bindingof the diagnostic reagent with phosphatidylserine on apoptotic cells. Insome instances there may not be a requirement for a reagent tofacilitate measurement or colocalization of the diagnostic reagent. Inthese particular instances, the accessory reagent can be, for example,the buffer in which the diagnostic reagent works optimally.

Additionally, an accessory reagent can be more than one reagent presentin the medium containing the diagnostic reagent. For example, anaccessory reagent can have two components such as a lysis reagent and acofactor or other agent which facilitates measurement of apoptoticactivity. Moreover, reagents which enhance the specificity of thediagnostic reagent can additionally be included as an accessory reagent.Dyes which measure cell viability are specific examples of suchaccessory reagents which enhance specificity. Those skilled in the artwill know or can determine what combinations of agents should be used asan accessory reagent. Thus, the choice of the accessory reagent willdepend on the diagnostic molecule and the apoptotic event to bemeasured. Those skilled in the art will similarly know which accessoryreagents are applicable with which diagnostic reagents given theteachings described herein.

Depending on the cell type, number and diagnostic reagent used,incubation time periods will vary according to the need and the specificapoptotic activity to be measured. For example, using Jurkat cells, andmeasuring for the induction of caspase activity as little as a 30 minuteincubation period with the diagnostic reagent is required for detectionof a signal. This period of incubation is measured after, for example,the induction of apoptosis. For the specific example, of caspaseactivity, enzyme production of product will increase over time and willsimilarly lead to an increase in signal. Therefore, longer incubationperiods with the diagnostic reagent will lead to stronger signals. Otherfactors can be adjusted to modify the incubation time needed todetermine the specific apoptotic activity of a cell. For example, cellnumber or reagent concentrations can be increased to decrease theincubation time necessary to obtain a reliable measurement. Suchmodifications are known to those skilled in the art or can be routinelydetermined by testing various cell numbers and reagent concentrationsover several time points to determine those modifications which provideoptimal performance for the particular need. Normally, not more than 6hours is required for detection of apoptotic activity, preferablybetween about 30 minutes and 2 hours and usually not more than about 1hour of incubation with a diagnostic reagent is necessary for measuringthe specific apoptotic activity of a cell.

As with cell type, cell number, reagent concentrations and incubationtimes, so can medium volumes be varied to obtain a desired result or tofit a particular need. For example, medium volumes sufficient to coverthe cell sample should be used so as to allow adequate measurement ofapoptotic activity. Volumes can be adjusted according to the solubilityproperties of the diagnostic reagent and the accessory reagent.Essentially, all that is necessary is for the medium to contain theappropriate amounts of required reagents and to make the reagentsavailable to the molecules to be measured. Preferably the volumes shouldbe confined to that of a microwell chamber such as a 96-well ELISAplate. Such volumes are generally between about 1 and 200 μl, morepreferably between about 30 and 125 μl and preferably about 100 μl.Those skilled in the art will know what volumes are useful for theparticular situation.

Thus, the method is amenable to a multiwell format assay where largenumbers of samples can be screened rapidly and efficiently. Inparticular, a 96 well format provides practical advantages since platesappropriate for manipulations and measuring devices are commerciallyavailable. Such procedures can be further automated to increase furtherthe speed and efficiency of the method. These features, combined withthe specificity of the method, allows for the high throughput screeningof compounds which either induce or inhibit apoptosis. For example, alibrary of test compounds can be administered to a plurality of cellsample populations and then assayed for their ability to induce orinhibit apoptosis. Each of the different test compounds is administeredfor a sufficient time so as to induce or inhibit apoptosis prior todetermining specific apoptosis activity. Such an incubation time isusually about 2 minutes to 4 hours. Identified compounds are valuablefor both therapeutic and diagnostic purposes since they can allow forthe treatment and detection of apoptotic mediated diseases. Suchcompounds are also valuable in research related to apoptotic mechanismssince they can help deduce further molecular events and provide furtherspecificity for the discovery and development of future compounds.

The single-well microscale method described previously for determiningthe specific apoptotic activity of a cell can also be employed for therapid screening and identification of inhibitors of apoptosis.Therefore, the invention also provides a rapid method of identifying acompound which inhibits apoptosis. The method consists of (a) separatelycontacting a plurality of cell populations with a different compound tobe tested for apoptotic inhibiting activity; (b) incubating said cellswith a direct stimulus of the cell death pathway for a period of betweenabout 2 minutes to 3 hours, and (c) measuring the specific apoptoticactivity of the cells.

The invention further provides a method of identifying a compound whichinduces apoptosis. The method consists of (a) providing a celloverexpressing a cell survival polypeptide, the cell survivalpolypeptide being overexpressed at a level which is sufficient toprevent the induction of apoptosis; (b) treating the cell overexpressingthe cell survival polypeptide with a direct stimulus of the cell deathpathway; (c) adding a compound to be tested for apoptotic inducingactivity, and (d) determining cellular apoptotic activity, wherein thepresence of apoptotic activity is indicative of the compound being anapoptotic inducer.

The method described above for the identification of inducers ofapoptosis is particularly valuable providing an enhanced format formeasuring apoptotic activity in that a cell is treated so that it is“poised” for programmed cell death. In this way the cell has synthesizedand/or activated all necessary components that are required forprogrammed cell death. All that is necessary is a stimulus to push thecell past its holding point and into apoptosis. A positive test compoundis just that stimulus to cause the cell to progress into programmed celldeath.

The holding point which prevents the cell from proceeding intoprogrammed cell death is the overexpression of a cell survivalpolypeptide. Cell survival polypeptides are characterized in that theyexhibit the ability to prevent apoptosis when expressed or activated ina cell induced to undergo apoptosis. For example, in the absence of afunctioning cell survival polypeptide, a cell treated with an apoptoticinducer will initiate the programmed cell death pathway and eventuallydie by apoptosis. However, in the presence of a cell survivalpolypeptide, treatment with an apoptotic inducer can initiate theprogrammed cell death pathway but the cell will survive due toinhibition of one or more events along the pathway. Depending upon thepoint at which the cell survival polypeptide functions, the programmedcell death pathway can be inhibited early or relatively late within theexecution of the cascade of events leading to ultimate cell death. Cellsurvival polypeptides and their encoding nucleic acids are well known inthe art and include, for example, the Bcl-2 family of related proteinsBcl-2, Bcl-xL, Mcl-1, E1B-19K as well as inhibitors of the caspases suchas p35, crmA and the dominant-negative forms of the caspases. Theseforms include, for example, caspase's with an inactivating mutation ofthe active site cysteine.

Overexpression of a cell survival polypeptide an be achieved using, forexample, recombinant methods known to those skilled in the art. Routineprocedures for performing such recombinant expression methods aredescribed in, for. example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992), andin Ansubel et al., Current Protocols in Molecular Biology, John Whileyand Sons, Baltimore, Md. (1989). Such methods can be used to expressstably or transiently a cell survival polypeptide at a level which issufficient to prevent the induction of apoptosis. The nucleic acidencoding the cell survival polypeptide can be encoded by, for example, ahomologous nucleic acid derived from the same species or cell type, oralternatively, it can be encoded by a heterologous nucleic acid derivedfrom a different species or cell type. The source of the encodingnucleic acid is not important so long as the encoded cell survivalpolypeptide exhibits apoptosis inhibiting activity.

A level of expression of a cell survival polypeptide which is sufficientto prevent the induction of apoptosis is known to those skilled in theart and can also be routinely determined by those skilled in the art.Expression vectors and systems are known and commercially availablewhich provide for recombinant polypeptide expression. It is a routinematter for one skilled in the art to choose a vector or system whichwill provide sufficient levels of expression in a particular host cell.Alternatively, the expression level sufficient to prevent the inductionof apoptosis can be routinely determined by expressing the cell survivalpolypeptide and then measuring whether the cell survives after treatmentwith an apoptotic stimulus.

In addition to recombinant methods of overexpressing a cell survivalpolypeptide, a cell can be used which inherently over expresses a cellsurvival polypeptide. A specific example of a cell inherentlyoverexpressing a cell survival polypeptide is the B cell lymphoma inwhich Bcl-2 was initially identified. This leukemia has a translocationof chromosome 14 to 18 causing high level expression of Bcl-2 andtherefore cell survival. The leukemic phenotype is due to the increasedcell survival. Other cell lines which inherently overexpress a cellsurvival polypeptide, either by natural or unnatural mechanisms, existand can similarly be used in the methods of the invention.

The block from apoptosis due to overexpression of a cell survivalpolypeptide and the treatment of the cells with a direct stimulus ofapoptosis provide antagonistic influences to the cell. In this way, thecells are then essentially poised for programmed cell death. A directstimulus for apoptosis can be a variety of different insults to the cellincluding, molecular, environmental and physical stimuli. As definedpreviously, such stimuli are known to those skilled in the art and canbe characterized by activating a molecule within the apoptotic pathway.Examples of direct stimuli of apoptosis include inducers such as Fasligand, anti-Fas antibody, Staurosporine, ultraviolet and gammairradiation. Thus, treatment of a cell over expressing a cell survivalpolypeptide with a direct stimulus of apoptosis will prime the cell forapoptosis since both positive and negative signals are providingbalancing effects. One advantage of this priming is that all cell deathcomponents are available for apoptosis once a signal is received thatoverrides the block of the cell survival polypeptide. This advantageallows for the rapid induction of apoptosis which can be beneficial whenused to screen for compounds that possess apoptosis inducing activitywhen Bcl-2 or Bcl-xL is the cell survival polypeptide. Such cells areparticularly useful in screening for inhibitors of Bcl-2 or Bcl-xL,respectively.

In addition to treating the cells with a direct stimulus of apoptosis,the cells are also treated with one or more compounds which are to betested for apoptosis inducing activity. The compounds can be, forexample, small molecules, peptides, polypeptides, proteins or othermacromolecules. Essentially, the type of compound which is to be testedis unimportant, only that the user desires to test whether the compoundhas apoptotic inducing activity. Therefore, the assay is applicable fora variety of different settings, including clinical, diagnostic and drugdiscovery.

Although essentially any method which distinguishes apoptosis fromnecrosis, for example, can be used to determine cellular apoptoticactivity, the use of the single-well method described previously formeasuring apoptotic activity provides advantages. This method allows forthe rapid and efficient determination of apoptosis in what can beutilized in a multiwell or high throughput format. Therefore, once thecompounds are administered to the cell, apoptotic activity of the cellis determined by, for example, the rapid measurement of caspaseactivity, or alternatively any of a variety of other methods known inthe art. Those samples which yield positive results compared to controlsamples are indicative of compounds which will induce programmed celldeath.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Measurement of Specific Apontotic Activity

This Example describes the use and characteristics of the single-wellmethod for the measurement of specific apoptotic activity and screeningof compound libraries for inducers of apoptosis.

A human T cell leukemia clonal (Jurkat) cell line was used below fordemonstrating and characterizing the single-well method for measuringspecific apoptotic activity of a cell. For the screening of compounds,this Jurkat cell line was stably transfected with an expressionconstruct for the cell survival polypeptide Bcl-2 and clonal cell lineswere generated from the stable transfectants. The cell lines wereconstructed using methods known in the art and essentially as describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1992). Cell lines were cultured in RPMI1640 and 10% fetal bovine serum (FBS), in the presence of a maintenancelevel of G418 (200 μg/ml). A neo-transfected cell line was constructedand cultured in a similar way for use as a control.

To ensure that the transfected cell lines were expressing Bcl-2, celllysates were prepared from positive clones and Bcl-2 levels weremeasured by polypeptide blot analysis. Briefly, cell lysates wereprepared from 500,000 transfectants by first pelleting the culture andthen solubilizing the cells in mercaptoethanol-SDS buffer (5% (w/v)sodium dodecyl sulphate, 15% (v/v) glycerol, 0.01% (w/v) bromophenolblue, 10% (v/v) β-mercaptoethanol in 250 mM Tris-HCl, pH6.0).Polypeptides within the lysate were separated by SDS-polyacrylamide gelelectrophoresis and transferred to nitrocellulose by electro-blotting.Detection was performed using a Bcl-2 specific monoclonal antibody thatrecognizes both endogenous and transfected forms of Bcl-2 (Hockenbery etal., Nature 348:334-336 (1990)). Expression levels were quantified byusing different amounts of purified recombinant Bcl-2 run on the samegel as a comparison. Comparison of the transfected Bcl-2 expressionlevels with the known amounts indicated an expression level of about 6ng/10⁶ cells. The expression level of endogenous Bax, detected with ananti-Bax specific antibody and quantified in a similar manner wasdetermined to be about 1 ng/10⁶ cells (Reed et al., Analytical Biochem.205:70-76 (1992)).

To induce apoptosis, either a neo-transfected Jurkat cell line or theabove Bcl-2 transfected cell line was used. Specifically, the cell lineswere harvested, washed in RPMI 1640 (without phenol red as an indicator)and 10% FBS, and resuspended at a density of 1.33×10⁶ cells/ml in thesame medium. Cells were plated in 96-well plates at 100,000 cells/wellin a 75 μl volume and allowed to acclimatise for 1 hour at 37° C.Apoptosis was induced by incubation with an anti-Fas antibody which wasadded to the cells to a final concentration of 100 ng/ml and allowed toincubate for 3 hours at 37° C. (11 μl of a 10× stock solution; MBL,Pavera: Madison, Wis.; Yonehara et al., Experimental Med. 169:1747-1756(1989)). Alternatively, for screening procedures, compounds werepre-incubated with the Bcl-2 transfected cells for 1 hour at 37° C.prior to the addition of anti-Fas antibody. Compounds to be tested wereadded to duplicate wells at a 4× concentration (25 μl) resulting in afinal concentration of 5 μg/ml compound and 1% DMSO.

Apoptosis was determined by measuring aspartic specific cysteineprotease (caspase) activity following anti-Fas antibody incubation.Briefly, the cells were lysed by the addition of 10× lysis buffer andshaking in an orbital shaker for 5 min at room temperature (12.3μl/well; 2 mM PMSF, 10 mM DTT, 10 μg/ml Pepstatin A, 10 μg/ml Leupeptin,50 μg/ml Aprotenin, 1 mM EDTA, 1 mM EGTA and 5% CHAPS in 10× hypotonicbuffer (100 mM Hepes pH 7.4, 420 mM KCl, 50 mM MgCl₂)). ICE buffer wassubsequently added (70 μl/well of a 2× stock prepared as follows: 40 mMHepes, pH 7.5, 2 mM EDTA, 20% Sucrose and 0.2% CHAPS to which DTT wasadded fresh before the start of every assay to a final concentration of10 mM) and the enzyme assay was initiated by addition of the DEVD-AMCsubstrate to a final 2 μM concentration (10 μl of a 20× stock).Alternatively, the lysis buffer, ICE buffer and DEVD-AMC (SEQ ID NO:1)substrate can be combined into a single buffer and added in one step.Substrate cleavage activity was measured at thirty minute intervals overa period of two hours beginning at time zero in a fluorescent platereader. The excitation wavelength was 360/40 nm and the emissionwavelength 460/40 nm.

Controls run on each multiwell plate included a matching neo-transfectedcell line treated with anti-Fas antibody, but no compound, as thepositive death control and Bcl-2 transfected cells with-and withoutantibody as the background controls.

The characterization and use of the above method for screening inducersof apoptosis is described further below.

Studies were initially carried out comparing the single-well methoddescribed above (where the entire experiment is carried out in the samewell), with a known method where cells are spun down and washed afteranti-Fas antibody treatment and before addition of lysis buffer. Thiscomparison is presented in FIG. 1A. The results show that both methodsyield comparable caspase activity as demonstrated by the CPP32-likeenzymatic cleavage of the DEVD-AMC (SEQ ID NO:1) substrate inneo-transformed cell lines. In contrast, cell lines transfected withBcl-2 are protected from Fas induced death and show little increase incleavage (FIG. 1B). These results demonstrate that the single-wellmethod can accurately measure both the induction and inhibition ofcaspase activity at a sensitivity comparable to previously used methods.

Further characterization of the single-well method demonstrated that thepresence of 10% FBS was beneficial throughout the course of the assay,up to lysis of the cells. For example, substitution with 0.5% bovineserum albumin resulted in induction of CPP32-like activity in theneo-transfected Jurkat cells in the absence of anti-Fas antibodytreatment. This effect is likely due to induction of apoptosis throughgrowth factor withdrawal. However, the expression of Bcl-2 was able toprotect the cells from this effect.

Further, compounds in libraries for high throughput screening areroutinely maintained as stock solutions dissolved in DMSO. Furthercharacterization of the single-well method was carried out to determinepermissible levels of DMSO permissible in the method. The resultsindicated that up to at least 1% DMSO had no significant effect on thecells or the accuracy of the method. Testing of the possible effect ofthe media component phenol red, which is-normally used as an indicatorin RPMI, had a slight, but significant, quenching effect on thefluorescence readings. Due to this effect, phenol red was removed fromthe media in all further assays.

To optimize the incubation time and concentration of anti-Fas antibody,studies were performed which measured the caspase activity over a rangeof incubation time points and concentration of anti-Fas antibody.Caspase activity was determined in both neo- and Bcl-2 transfectedcells. In the neo-transfected cells, induction of a CPP32-like activityincreased with anti-Fas antibody concentration up to 30 ng/ml, afterwhich a plateau was reached. A slight, but significant, increase inCPP32-like activity was observed in the Bcl-2-transfected Jurkats at anantibody concentration of 100 ng/ml.

At anti-Fas antibody concentrations above 10 ng/ml the highest enzymaticactivity was observed between 2 and 5 hours after addition of theantibody to neo-transfected Jurkat cells. Longer incubation timesresulted in a decrease of observable CPP32-like activity (FIG. 2A). InBcl-2 transfected cells, what little enzymatic activity was detected wasseen only at an incubation time of 4 hours (FIG. 2B). From the aboveresults an antibody concentration of 100 ng/ml incubated for 3 hours,was chosen as the standard running conditions for the assay.

Further characterization of the assay was performed by assessing boththe intra-assay and inter-assay variation. Specifically, the intra-assayvariation of the procedure was examined by running 12 wells of each ofthe neo-transfected and of the Bcl-2 transfected cell lines on the sameplate at the same time in the presence or absence of anti-Fas antibody.The results of this study are shown in Table 1 and reveal an overallintra-assay variation of 6.8% or less of the mean of the caspaseactivity of the positive, neo-transfected control treated with anti-Fas.

TABLE 1 Intra-assay variation (RFU) Mean Std Dev % CV Neo-transfected +anti-Fas antibody 228.3 15.6 6.8 Neo-transfected − anti-Fas antibody17.7 4.3 1.9 Bcl2-transfected + anti-Fas antibody 10.2 3.3 1.4Bcl2-transfected − anti-Fas antibody 2.4 3.5 1.5 RFU = RelativeFluorescent Units Std Dev = Standard Deviation % CV = Percentcoefficient of Variation Percent coefficient of Variation is defined as:% CV = (Standard Deviation/Mean of Neo-transfected + anti-Fas) *100

Alternatively, the inter-assay variation was determined by running eachof the neo- or Bcl-2 transfected cell lines 24 on separate plates overthe course of two days. Each plate was set up individually.

The result of this determination are presented in Table 2 and reveal anoverall inter-assay variation of 7.0% or less of the mean of the caspaseactivity of the positive, neo-transfected control treated with anti-Fas.

TABLE 2 Intra-assay variation (RFU) Mean Std Dev % CV Neo-transfected +anti-Fas antibody 221.5 15.5 7.0 Neo-transfected − anti-Fas antibody 7.72.9 1.3 Bcl2-transfected + anti-Fas antibody 22.4 8.2 3.7Bcl2-transfected − anti-Fas antibody −1.5 2.0 0.9 RFU = RelativeFluorescent Units Std Dev = Standard Deviation % CV = Percentcoefficient of Variation Percent coefficient of Variation is defined as:% CV = (Standard Deviation/Mean of Neo-transfected + anti-Fas) *100

EXAMPLE II Screening Compounds for the Induction of Apoptosis

This Example describes the use of the screening of a compound libraryfor the detection of apoptotic inducers.

The single-well method for determining specific apoptotic activity wasused in conjunction with Bcl-2 transfected cells to identify compoundsthat exhibit apoptotic inducing activity. The procedures and the Bcl-2transfected cell line was described above in Example I.

Briefly, 960 compounds chosen from a chemical library were examined fortheir ability to induce apoptosis in cells that were primed by theincubation of Bcl-2 stably transfected cells with anti-Fas antibody. Thescreening was performed in a multiwell or high throughput mode and thecompounds were incubated at a concentration of 5 μg/ml. The results ofthis screen are shown in FIG. 3. Values are expressed as a percentage ofcaspase activity of the positive, neo-transfected control treated withanti-Fas present on each plate. The levels of caspase activity rangedfrom 62.1 to −11.5% of the non-Bcl-2 protected control. A mean value of1.7% and a standard deviation of 4.2% was also observed.

The threshold for defining a compound as positive was set at 24% of thecaspase activity of neo-transfectants in the absent of any compound (46relative fluorescence units). This number was determined by the meanplus 3 standard deviations of the negative controls (anti-Fas treatedBcl-2-transfected cells) shown in Table 2 for calculating theinter-assay variation. This threshold is valid assuming a normaldistribution for the results. Given this assumption, the probabilitythat a negative compound will exceed the mean of the negative controlsby more than 3 standard deviations is 0.0028. Using this criterion, 5positives were identified from the 960 compounds screened.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to describe morefully the state of the art to which this invention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

4 1 4 PRT Artificial Sequence Description of Artificial SequenceSubstrate analogue of caspase family member. Apoptotic specificdiagnostic reagent. 1 Asp Glu Val Asp 1 2 4 PRT Artificial SequenceDescription of Artificial Sequence Substrate analogue of caspase familymember. Apoptotic specific diagnostic reagent. 2 Tyr Val Ala Asp 1 3 5PRT Unknown Description of Unknown Organism Conserved pentapeptidesequence within active site of known caspases 3 Gln Ala Cys Arg Gly 1 54 5 PRT Unknown Description of Unknown Organism Conserved pentapeptidesequence within active site of known caspases 4 Gln Ala Cys Gly Gly 1 5

What is claimed is:
 1. A rapid, single-well method of identifying acompound which inhibits apoptosis comprising: (a) separately contactinga plurality of cell populations with a different compound to be testedfor apoptotic inhibiting activity; (b) incubating said cell populationswith a direct stimulus of the cell death pathway for a period of betweenabout 2 minutes to 3 hours; and (c) measuring the specific apoptoticactivity of the cell populations, wherein inhibition of the specificapoptotic activity is indicative that said compound is an inhibitor ofapoptosis.
 2. The method of claim 1, wherein said direct stimulus of thecell death pathway is selected from the group consisting of Fas ligand,anti-Fas antibody, staurosporine, UV and gamma irradiation.
 3. Themethod of claim 1, wherein step (c) further comprises lysing said cellpopulation and determining caspase activity in said lysate.
 4. Themethod of claim 1, wherein said compound exhibits caspase inhibitoryactivity.
 5. The method of claim 1, wherein said compound inhibitsapoptosis by promoting the activity of a cell survival polypeptide. 6.The method of claim 1, wherein said compound exhibits cell deathpolypeptide inhibitory activity.